B.t. is a gram-positive soil bacterium which produces crystal proteins during sporulation which are specifically toxic to certain orders and species of insects. Many different strains of B.t. have been shown to produce insecticidal crystal proteins. Compositions including B.t. strains which produce insecticidal proteins have been commercially available and used as environmentally acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
A number of genes encoding crystal proteins have been cloned from several strains of B.t. A good overview is set forth in H. Hofte et al., Microbiol. Rev., 53, pp. 242-255 (1989). While this reference is not prior art with respect to the present invention, it provides a good overview of the genes and proteins obtained from B.t. and their uses, a nomenclature and classification scheme, and has an extensive bibliography.
Also see A. R. Aronson, et al., Microbiol. Rev., 50, pp. 1-24 (1986) for an earlier review of work relating to the insecticidal activity of B.t..
The B.t. crystal protein is active in the insect only after ingestion. After ingestion by a lepidopteran insect, the alkaline pH and proteolytic enzymes in the mid-gut solubilize the crystal allowing the release of the toxic components. These toxic components poison the mid-gut cells causing the insect to cease feeding and, eventually, to die. In fact, B.t. has proven to be an effective and environmentally safe insecticide in dealing with lepidopteran pests.
One predominant class of toxin crystal proteins produced by many of the B.t. strains is known as the P-1 type of proteins (and more recently as the CryI type of proteins). The CryI proteins have molecular masses of about 130,000 Daltons (Da). The genes for the CryI crystal proteins as well as those of other crystal protein genes have been discovered to reside on large plasmids that occur naturally in B.t.
The present invention is a result of developments by the inventor, building on his experience with other B.t. genes and proteins. The inventor has isolated and purified a gene identified as cryIIA (previously referred to as the "P-2," "cryB1" or the "cryBI" gene) and the encoded CryIIA protein (previously referred to variously as the "P2 protein," "P-2 toxin," "P-2 delta-endotoxin" or "CryB1" protein) resulting from the cryIIA gene expression. These are disclosed and claimed in the Parent Application. The cryIIA gene, obtained from B.t. var. kurstaki (hereinafter "B.t.k.") strain HD-263 is also described in W. P. Donovan et al., J. Biol. Chem., 263, PP. 561-567 (1988) (hereinafter "Donovan (1)"), with a correction to the nucleotide sequence of the cryIIA gene and the amino acid sequence of the CryIIA protein published in W. P. Donovan, et al., J. Biol. Chem., 264, p. 4740 (1989) (hereinafter "Donovan (2)"). The CryIIA gene contains 633 codons and encodes a CryIIA protein having a molecular mass of 70,860 Da. As reported in Donovan (1) and in T. Yamamoto et al., Biochem. Biophys. Res. Commun., 103, pp. 414-421 (1981), the CryIIA protein is toxic to both lepidopteran (caterpillars) and dipteran (mosquitos) insects.
In connection with the work relating to the cryIIA gene and CryIIA protein, the inventor discovered that B.t.k. contains a nucleotide sequence related to the cryIIA gene which was designated cryIIB (previously referred to as the "cryBI-related" sequence or "cryBII" sequence). The cryIIB gene has 633 codons and encodes a protein of 70,749 Da, the CryIIB protein (previously referred to as "CryB2"). Using the nucleotide sequence comparison program of C. Queen et al., Nucleic Acids Res., 12, pp. 581-599 (1984), it was determined that the protein coding region of 1,899 nucleotides of the cryIIB gene contained 89% positional identity with the protein coding region of 1,899 nucleotides of the cryIIA gene. Additionally, 557 out of 633 of the amino acids in the CryIIB protein were positionally identical to the corresponding amino acids in the CryIIA protein (88%). While the CryIIA and CryIIB proteins appear to be similar, measurement of their insecticidal activities indicated a substantially different insect toxicity between the two proteins. Thus, the proteins are related, but they clearly differ in their amino acid sequences and in their insecticidal activities.
The cryIIB gene and CryIIB protein were also investigated independently by other researchers as reported in W. R. Widner et al., J. Bacteriol, 171, pp. 965-974 (1989), published less than one year before the filing of the present application. Widner et al. (1989) reported a nucleotide sequence for the cryIIA gene (referred to in the article as a "cryB1" gene) and a cryIIB gene (referred to in the article as a "cryB2" gene). The nucleotide sequence for the cryB2 gene reported in Widner et al. (1989) is identical to the protein coding region of the cryIIB gene of the present invention with the exception of a translationally silent difference at nucleotide 1035 which the present inventor has found to be thymine, but which Widner et al. (1989) report as cytosine.
As explained hereinafter, the inventor also determined that the cryIIB gene did not express well with its native promoter, such that the CryIIB protein was produced in minimal amounts, if at all, in the native B.t. strains. Accordingly, as part of the present invention, the inventor created a recombinant hybrid fusion gene in which the promoter from the cryIIIA gene was fused to the protein coding region of the cryIIB gene. This resulted in substantially enhanced production of the CryIIB protein.
The cryIIIA gene and its product, the CryIIIA protein (referred to previously as the "cryC" gene and "CryC" protein) are described in W. P. Donovan et al., Mol. Gen. Genet., 214, pp. 365-372 (1988) (hereinafter "Donovan (3)"). The cryIIIA gene was isolated from a new strain of B.t. (designated EG2158) toxic to larva of Lepinotarsa decemlineata (Colorado potato beetle). The cryIIIA gene isolated by the inventor apparently is identical to a gene of B.t. var. tenebrionis, as reported by V. Sekar, et al., Proc. Natl. Acad. Sci. USA, 84, pp.7036-7040 (1987), and by H. Hofte, et al., Nucleic Acids Res., 15, p. 7183 (1987), as well as to a gene isolated from B.t. var. san diego, reported by C. Herrnstadt, et al., Gene, 57, pp. 37-46 (1987). B.t. strain EG2158 differs in several ways from B.t. tenebrionis and B.t. san diego.